7. PROJECT SUMMARY Five-Dimensional Single-Molecule Nanoscopy for Sensing and Imaging the Dynamic Functions of Biomolecules This project will implement five-dimensional (5D) single-molecule (SM) nanoscopy to detect and visualize the dynamics of biological structures within living cells with nanoscale resolution. This project will be the first demonstration of super-resolution (SR) fluorescence imaging capable of resolving the 3D position and 2D orientation (i.e., x, y, z, pitch, and yaw) of single molecules in living biological systems. Five-dimensional measurements are needed to elucidate biomolecular interactions because molecules are not simple isotropic spheres; their orientation and/or conformation critically determine how they interact with each other. Super-resolved fluorescence microscopy, awarded the Nobel Prize in Chemistry 2014 and also termed optical nanoscopy, produces images of structures within living cells with resolution beyond the optical diffraction limit (~250 nm for visible light). One fundamental drawback of SR microscopy is its inability to measure the activity and function of molecules (e.g., binding, conformation, structural disorder, etc.) since its images only depict the 2D or 3D spatial positions of fluorescent tags. This limitation is a consequence of traditional microscope designs, which cannot measure the phase or polarization of light. Here, 5D SM nanoscopy will be developed to measure the dynamic activities of biomolecules by innovating and combining two synergistic approaches: 1) use binding- activated fluorogenic probes for imaging biological structures and 2) design and utilize integrated optical hardware and image analysis software for visualizing the 3D position (x, y, z) and 2D orientation (? and ? in spherical coordinates) of fluorescent probes. Optical nanoscopes will no longer simply focus light onto a camera to create 2D images; rather, the fluorescent light within the imaging system will be ?bent? specifically so that molecular position and orientation can be directly measured from the images captured by the camera. Lipid nanodomains are thought to control the trafficking of biomolecules across the cell membrane. A critical barrier to understanding these activities is our inability to directly visualize these domains. Five-dimensional SM nanoscopy will visualize nano-polarity and nano-fluidity of cell membranes by using fluorescent molecular sensors to diffuse, collide, and temporarily bind to biomolecules of interest within a cell, lighting up in the process. The rotational mobility of these probes will directly measure the polarity and/or fluidity of their environment. The aggregation of A?1-42 on the membranes of cultured human neuroblastoma cells (SH-EP cells) will be studied with a two-color variant of 5D SM nanoscopy to obtain nanoscale resolution. Simultaneously, lipid rafts will be visualized using lipid-specific fluorescent molecular sensors. This approach will reveal the dynamic nanoscale interactions between lipid nanodomains and A?1-42, especially how lipid phase affects the binding of A?1-42 and how the accumulation of A?1-42 remodels membrane morphology. By visualizing the nanoscale dynamics of both biomolecules simultaneously with SM sensitivity, the formation mechanism of toxic A? species and the impact of membrane physiology on the progression of Alzheimer?s Disease will be elucidated.